Manipulation of transposable element families within, or derived from, the maize genome is a proven technique for the isolation of plant genes (see Chandler (1994) in The Maize Handbook, ed. Freeling and Walbot (Springer-Verlag, New York), pp. 647-652). The maize transposable element family, Mutator, has one of the highest forward mutation rates known in nature; these elements exhibit a high frequency of germinal insertion, an apparent bias for insertion into transcribed regions, and frequent, late, somatic excision. Mutator is represented by six classes of member elements whose DNA sequences are known, and so-called active Mutator lines depend upon the presence and expression of the autonomous regulatory element Mu-DR (Robertson (1978) Mutation Res. 51:21-28; Chandler and Hardeman (1992) in Adv. Genet., 30:77-122). The sequence conservation of the 220 base pair terminal-inverted-repeat DNA (TIR) of these elements has been exploited for PCR-based selection and functional analysis of insertion mutants for known DNA sequences, as embodied by the Trait Utility System for Corn (TUSC) program. While TUSC is designed for reverse genetics, a similar scheme assists cloning transposon-tagged genes in forward genetics fashion.
Forward gene tagging with Mutator is initiated by creating genetic crosses between donor Mu-active and recipient inactive lines. Mu-donor lines contain the Mu-DR regulatory element (Chomet et al. (1991) Genetics 122:447457) and high copy number of Mu elements, whereas the recipient lines are inbred lines having no active Mu elements. The Mu-donor line is usually chosen as the male parent because a greater number of mutagenized gametes can be sampled through the pollen. Those germinal insertion events that disrupt gene function predominantly behave as recessive mutations, so mutant phenotypes are most often discovered by observing the F2 progeny of the donor x recipient cross.
Once a phenotype of interest is noted, cosegregation analysis is performed within the mutant family. DNA is isolated from mutant individuals and subjected to restriction enzyme digestion and Southern analysis. Radiolabeled probes for each Mu element class are hybridized in-succession to the Southern blot to search for Mu-containing restriction fragments that cosegregate with the phenotype in question. A subgenomic library is then prepared by excising the cosegregating fragment from an agarose gel and cloning into a suitable vector. Positive clones must be identified with the appropriate Mu probe; each candidate must then be characterized by restriction mapping, subcloning, sequence analysis, and confirmation. Amplified Fragment Length Polymorphisms (AFLP) technology has been designed to generate large numbers of randomly distributed molecular markers (see, for example, European Patent Application No. 0534858 A1).
Despite the elegant genetic and molecular methods involved in transposon tagging, the field and laboratory processes involved are frequently protracted and very labor intensive. A more efficient method for rapid isolation and identification of transposable element-tagged genes is needed. The present invention provides this and other advantages.